Newly generated interspecific wine yeast hybrids introduce flavour and aroma diversity to wines

Increasingly, winemakers are looking for ways to introduce aroma and flavour diversity to their wines as a means of improving style and increasing product differentiation. While currently available commercial yeast strains produce consistently sound fermentations, there are indications that sensory complexity and improved palate structure are obtained when other species of yeast are active during fermentation. In this study, we explore a strategy to increase the impact of non-Saccharomyces cerevisiae inputs without the risks associated with spontaneous fermentations, through generating interspecific hybrids between a S. cerevisiae wine strain and a second species. For our experiments, we used rare mating to produce hybrids between S. cerevisiae and other closely related yeast of the Saccharomyces sensu stricto complex. These hybrid yeast strains display desirable properties of both parents and produce wines with concentrations of aromatic fermentation products that are different to what is found in wine made using the commercial wine yeast parent. Our results demonstrate, for the first time, that the introduction of genetic material from a non-S. cerevisiae parent into a wine yeast background can impact favourably on the wine flavour and aroma profile of a commercial S. cerevisiae wine yeast.     

Generation of intra- and interspecific Saccharomyces hybrids with improved oenological and aromatic properties

Non-wine yeasts could enhance the aroma and organoleptic profile of wines. However, compared to wine strains, they have specific intolerances to winemaking conditions. To solve this problem, we generated intra- and interspecific hybrids using a non-GMO technique (rare-mating) in which non-wine strains of S. uvarum, S. kudriavzevii and S. cerevisiae species were crossed with a wine S. cerevisiae yeast. The hybrid that inherited the wine yeast mitochondrial showed better fermentation capacities, whereas hybrids carrying the non-wine strain mitotype reduced ethanol levels and increased glycerol, 2,3-butanediol and organic acid production. Moreover, all the hybrids produced several fruity and floral aromas compared to the wine yeast: β-phenylethyl acetate, isobutyl acetate, γ-octalactone, ethyl cinnamate in both varietal wines. Sc × Sk crosses produced three- to sixfold higher polyfunctional mercaptans, 4-mercapto-4-methylpentan-2-one (4MMP) and 3-mercaptohexanol (3MH). We proposed that the exceptional 3MH release observed in an S. cerevisiae × S. kudriavzevii hybrid was due to the cleavage of the non-volatile glutathione precursor (Glt-3MH) to detoxify the cell from the presence of methylglyoxal, a compound related to the high glycerol yield reached by this hybrid. In conclusion, hybrid generation allows us to obtain aromatically improved yeasts concerning their wine parent. In addition, they reduced ethanol and increased organic acids yields, which counteracts climate change effect on grapes.     

MET17 and hydrogen sulfide formation in Saccharomyces cerevisiae

Commercial isolates of Saccharomyces cerevisiae differ in the production of hydrogen sulfide (H(2)S) during fermentation, which has been attributed to variation in the ability to incorporate reduced sulfur into organic compounds. We transformed two commercial strains (UCD522 and UCD713) with a plasmid overexpressing the MET17 gene, which encodes the bifunctional O-acetylserine/O-acetylhomoserine sulfhydrylase (OAS/OAH SHLase), to test the hypothesis that the level of activity of this enzyme limits reduced sulfur incorporation, leading to H(2)S release. Overexpression of MET17 resulted in a 10- to 70-fold increase in OAS/OAH SHLase activity in UCD522 but had no impact on the level of H(2)S produced. In contrast, OAS/OAH SHLase activity was not as highly expressed in transformants of UCD713 (0.5- to 10-fold) but resulted in greatly reduced H(2)S formation. Overexpression of OAS/OAH SHLase activity was greater in UCD713 when grown under low-nitrogen conditions, but the impact on reduction of H(2)S was greater under high-nitrogen conditions. Thus, there was not a good correlation between the level of enzyme activity and H(2)S production. We measured cellular levels of cysteine to determine the impact of overexpression of OAS/OAH SHLase activity on sulfur incorporation. While Met17p activity was not correlated with increased cysteine production, conditions that led to elevated cytoplasmic levels of cysteine also reduced H(2)S formation. Our data do not support the simple hypothesis that variation in OAS/OAH SHLase activity is correlated with H(2)S production and release.     

Improvement of pectinase production by interspecific hybrids of Aspergillus strains

Protoplast fusion induced by polyethylene glycol and Ca²⁺, was performed between auxotrophic mutants of pectinolytic fungi Aspergillus sp. CH-Y-1043 (A13) ade ⁻ and Aspergillus flavipes ATCC-16795 (F7) lys ⁻. Prototrophic colonies were developed on minimal medium with a fusion frequency of 1·0×10⁻². The reversion frequency of the mutation in spores and protoplasts was low and ranged from 2·0 to 4·0×10⁻⁷. Four prototrophic hybrids (HH, HE, HF and HJ) exhibited enhanced production of endo-pectinase and pectin-lyase. The highest production was observed in HJ ; maximum activities were 150 and 160% respectively, whereas the exo-pectinase production was similar to the wild-type strain Aspergillus sp. CH-Y-1043. Hybrid HJ showed the greatest growth ; nevertheless, specific endo-pectinase and pectin-lyase activities were higher in all hybrids than those produced by the wild-type strains.     

Enhanced diversity and aflatoxigenicity in interspecific hybrids of Aspergillus flavus and Aspergillus parasiticus

Aspergillus flavus and A. parasiticus are the two most important aflatoxin‐producing fungi responsible for the contamination of agricultural commodities worldwide. Both species are heterothallic and undergo sexual reproduction in laboratory crosses. Here we examine the possibility of interspecific matings between A. flavus and A. parasiticus. These species can be distinguished morphologically and genetically, as well as by their mycotoxin profiles. Aspergillus flavus produces both B aflatoxins and cyclopiazonic acid (CPA), B aflatoxins or CPA alone, or neither mycotoxin; Aspergillus parasiticus produces B and G aflatoxins or the aflatoxin precursor O‐methylsterigmatocystin, but not CPA. Only four of forty‐five attempted interspecific crosses between opposite mating types of A. flavus and A. parasiticus were fertile and produced viable ascospores. Single ascospore strains from each cross were shown to be recombinant hybrids using multilocus genotyping and array comparative genome hybridization. Conidia of parents and their hybrid progeny were haploid and predominantly monokaryons and dikaryons based on flow cytometry. Multilocus phylogenetic inference showed that experimental hybrid progeny were grouped with naturally occurring A. flavus L strain and A. parasiticus. Higher total aflatoxin concentrations in some F1 progeny strains compared to midpoint parent aflatoxin levels indicate synergism in aflatoxin production; moreover, three progeny strains synthesized G aflatoxins that were not produced by the parents, and there was evidence of allopolyploidization in one strain. These results suggest that hybridization is an important diversifying force resulting in the genesis of novel toxin profiles in these agriculturally important fungi.     

Genetic control of hydrogen sulfide retention in Saccharomyces cerevisiae

Summary The ability to retain hydrogen sulfide (H₂S) inSaccharomyces cerevisiae is under nuclear gene control. Mutants with the ability to retain greater amounts of H₂S than the parent have been isolated and characterised.     

Endogenous production of hydrogen sulfide in mammals

Summary. Hydrogen sulfide is one of three gases involved in biological functions and synthesized in vivo. Like NO and CO, it seems to act as a neuromodulator: it modulates NMDA glutamate receptor function. CBS seems to be the only source of hydrogen sulfide in the brain, whereas the liver synthesizes hydrogen sulfide via cystathionase. In the heart, the third pathway for the hydrogen sulfide synthesis, the 3-mercaptopyruvate pathway is used. Only two diseases characterized by alterations of hydrogen sulfide metabolism have been described: decreased hydrogen sulfide synthesis in the brains of Alzheimers disease patients and increased hydrogen sulfide synthesis due to the overexpression of CBS in Down syndrome patients.     

The role of amino acids in the regulation of hydrogen sulfide production during ultradian respiratory oscillation of Saccharomyces cerevisiae

We previously demonstrated that periodic H2S production during aerobic continuous culture of Saccharomyces cerevisiae resulted in ultradian respiratory oscillation, and that H2S production was dependent on the activity of sulfate uptake and the level of sulfite. To investigate the mechanism of regulation of the sulfate assimilation pathway and of respiratory oscillation, several amino acids were pulse-injected into cultures during respiratory oscillation. Injection of sulfur amino acids or their derivatives perturbed respiratory oscillation, with changes in the H2S production profile. Four major regulators of H2S production in the sulfate assimilation pathway and respiratory oscillation were identified: (1) O-acetylhomoserine, not O-acetylserine, as a sulfide acceptor, (2) homoserine/threonine as a regulator of O-acetylhomoserine supply, (3) methionine/S-adenosyl methionine as a negative regulator of sulfate assimilation, and (4) cysteine (or its derivatives) as an essential regulator. The results obtained after the addition of DL-propargylglycine (5 microM and 100 microM) and cystathionine (50 microM) suggested that the intracellular cysteine level and cystathionine gamma-lyase, rather than methionine/S-adenosylmethionine, play an essential role in the regulation of sulfate assimilation and respiratory oscillation. Based on these results and those of our previous reports, we propose that periodic depletion of cysteine (or its derivatives), which is involved in the detoxification of toxic materials originating from respiration, causes periodic H2S production.     

Endogenous production of hydrogen sulfide in isolated bovine eye

Hydrogen sulfide (H₂S) is a novel gasotransmitter with physiological and pathological functions in vascular homeostasis, cardiovascular system and central nervous system. In the present study, we determined the endogenous levels of H₂S in various tissues of the bovine eye. We also examined the basal levels of H₂S in response to donors (sodium hydrosulfide, NaHS and sodium sulfide, Na₂S), substrate (l-cysteine), inhibitors (propargylglycine, PAG and aminooxyacetic acid, AOA) and activator (S-adenosyl-l-methionine, SAM) of this gas in the bovine retina. H₂S was measured using a well established spectrophotometric method. The highest concentration of endogenous H₂S was detected in cornea (19 ± 2.85 nmoles/mg protein, n = 6) and retina (17 ± 2.1 nmoles/mg protein, n = 6). Interestingly, H₂S was not present in vitreous humor. The inhibitors of CSE and CBS; PAG (1 mM) and AOA (1 mM), significantly attenuated the production of H₂S in the bovine retina by 56.8 and 42%, respectively. On the other hand the activator of CBS; SAM (100 μM), H₂S donors; NaHS (1 μM) and Na₂S (100 μM), significantly increased endogenous levels of H₂S in bovine retina. l-cysteine (10–300 μM) produced a significant (P < 0.05) concentration-dependent increase in H₂S levels reaching a maximal at 300 μM. We conclude that H₂S is endogenously produced in various tissues of the isolated bovine eye. Moreover, endogenous levels of H₂S are enhanced in the presence of substrate (l-cysteine), an activator of CBS (SAM) and H₂S donors but are blocked by inhibitors of enzymes that synthesize this gas in neural retina.     

Contribution of cysteine aminotransferase and mercaptopyruvate sulfurtransferase to hydrogen sulfide production in peripheral neurons

Hydrogen sulfide (H₂S) is a gaseous neuromodulator produced from L‐cysteine. H₂S is generated by three distinct enzymatic pathways mediated by cystathionine γ‐lyase (CSE), cystathionine β‐synthase (CBS), and mercaptopyruvate sulfurtransferase (MPST) coupled with cysteine aminotransferase (CAT). This study investigated the relative contributions of these three pathways to H₂S production in PC12 cells (rat pheochromocytoma‐derived cells) and the rat dorsal root ganglion. CBS, CAT, and MPST, but not CSE, were expressed in the cells and tissues, and appreciable amounts of H₂S were produced from L‐cysteine in the presence of α‐ketoglutarate, together with dithiothreitol. The production of H₂S was inhibited by a CAT inhibitor (aminooxyacetic acid), competitive CAT substrates (L‐aspartate and oxaloacetate), and RNA interference (RNAi) against MPST. Immunocytochemistry revealed a mitochondrial localization of MPST in PC12 cells and dorsal root ganglion neurons, and the amount of H₂S produced by CAT/MPST at pH 8.0, a physiological mitochondrial matrix pH, was comparable to that produced by CSE and CBS in the liver and the brain, respectively. Furthermore, H₂S production was markedly increased by alkalization. These results indicate that CAT and MPST are primarily responsible for H₂S production in peripheral neurons, and that the regulation of mitochondrial metabolism may influence neuronal H₂S generation.

     

Polarographic measurement of hydrogen sulfide production and consumption by mammalian tissues

The role of nitric oxide (NO) in redox cell signaling is widely accepted. However, the biological role of another candidate small inorganic signaling molecule and the subject of this study, hydrogen sulfide (H2S), is much less known. H2S as a reductant and nucleophile has numerous potential cellular targets; however, its rapid biological oxidation suggests a fleeting cellular existence. The challenge of accurate real-time measurement of H2S at low micromolar or nanomolar concentrations in biological preparations represents a major impediment to H2S investigations. We here demonstrate the use of a novel polarographic H2S sensor (PHSS) to follow rapid changes in H2S concentration in common buffered biological solutions with a detection limit near 10 nM. The PHSS, used in combination with O2 and NO sensors in multisensor respirometry, shows stability, a high signal-to-noise ratio, and signal specificity for H2S. Preparations of rat vascular tissue exhibit H2S production on the addition of sulfhydryl-bearing amino acid substrates and H2S consumption when supplied with exogenous H2S. Taken together, these findings suggest the existence of dynamic steady-state cellular H2S levels. The PHSS should facilitate the investigation of H2S biology by providing a previously unattainable continuous record of H2S under biologically relevant conditions.     

Impact of substrates acclimation strategy on simultaneous biodegradation of hydrogen sulfide and ammonia

Three columns were differentiated with feeding mixture of H₂S and NH₃ (MFC), feeding NH₃ followed by H₂S (NFC), and feeding H₂S followed by NH₃ (SFC). Removal performance, biodegradation capacity and microbial community structures in the three columns were compared. The results show that NFC has a shorter acclimation period for the removal of NH₃ gas and nitrification than MFC. Under the high loading of H₂S and NH₃ at 164 and 82 g m⁻³ h⁻¹, respectively, NFC exhibited high removal efficiency of NH₃ (>95%) while the removal efficiencies were obtained at 63 and 75% in MFC and SFC, respectively. The removal of NH₃ gas in NFC was significantly attributed to nitrification (over 50%), while adsorption and chemical reaction contributed to the removal of NH₃ in MFC and SFC. The different biodegradation capacities of NH₃ could be due to the dissimilarity in the microbial population presented in each column.     

MET2 affects production of hydrogen sulfide during wine fermentation

The production of hydrogen sulfide (H₂S) during yeast fermentation contributes negatively to wine aroma. We have mapped naturally occurring mutations in commercial wine strains that affect production of H₂S. A dominant R₃₁₀G mutant allele of MET2, which encodes homoserine O-acetyltransferase, is present in several wine yeast strains as well as in the main lab strain S288c. Reciprocal hemizygosity and allele swap experiments demonstrated that the MET2 _R310G allele confers reduced H₂S production. Mutations were also identified in genes encoding the two subunits of sulfite reductase, MET5 and MET10, which were associated with reduced H₂S production. The most severe of these, an allele of MET10, showed five additional phenotypes: reduced growth rate on sulfate, elevated secretion of sulfite, and reduced production in wine of three volatile sulfur compounds: methionol, carbon disulfide and methylthioacetate. Alleles of MET5 and MET10, but not MET2, affected H₂S production measured by colour assays on BiGGY indicator agar, but MET2 effects were seen when bismuth was added to agar plates made with Sauvignon blanc grape juice. Collectively, the data are consistent with the hypothesis that H₂S production during wine fermentation results predominantly from enzyme activity in the sulfur assimilation pathway. Lower H₂S production results from mutations that reduce the activity of sulfite reductase, the enzyme that produces H₂S, or that increase the activity of l-homoserine-O-acetyltransferase, which produces substrate for the next step in the sulfur assimilation pathway.     

Genetic regulation of CFA expression in interspecific avian hybrids and somatic cell hybrids

Chicken fetal-leukemic antigen (CFA) is an oncodevelopmental antigen present on embryonic and neonatal chicken peripheral red blood cells (RBCs) but is not restricted to fetal stages of development in other avian species. Crosses between white Leghorn chickens and Japanese quail resulted in adult hybrids whose peripheral RBCs were positive for CFA. Of the four CFA determinants normally found in adult quail RBCs, only two were present on quail-chicken hybrid RBCs. Adult quail--chicken hybrid RBCs also possessed on CFA determinant associated with early development in both quail and chicken and one chicken-specific CFA determinant. Evidence is presented for the possible association of CFA-positive adult peripheral RBCs and the level of circulating reticulocytes. Crosses between pheasant and turkey (both with CFA-positive adult RBCs) resulted in hybrid adult RBCs expressing only a portion of the parental CFA determinants. Through the formation of somatic cell hybrids between adult chicken and embryonic Japanese quail RBCs, it was possible to induce the appearance of CFA determinants normally restricted to embryonic chicken RBCs. Approximately 50% of the hybrid cells showed reexpression of CFA, and this induction was both time and temperature dependent. Hybridization between RBCs of adult chicken and those of either adult Japanese quail or adult turkey failed to elicit the reexpression of chicken-specific CFA.     

The phs gene and hydrogen sulfide production by Salmonella typhimurium.

Salmonella typhimurium produces H2S from thiosulfate or sulfite. The respective pathways for the two reductions must be distinct as mutants carrying motations in phs, chlA, and menB reduced sulfite, but not thiosulfate, to H2S, and glucose repressed the production of H2S from thiosulfate while it stimulated its production from sulfite. The phs and chlA mutants also lacked a methyl viologen-linked thiosulfate reductase activity present in anaerobically grown wild-type cultures. A number of hydroxylamine, transposon Tn10 insertion, and Mu d1(Apr lac) operon fusion mutants defective in phs were characterized. One of the hydroxylamine mutants was an amber mutant, as indicated by suppression of its mutation in a supD background. The temperature-sensitive phs mutants produced H2S and methyl viologen-linked thiosulfate reductase at 30 degrees C but not at 42 degrees C. The reductases in all such mutants grown at 30 degrees C were as thermostable as the wild-type enzyme and did not differ in electrophoretic relative mobility, suggesting that phs is not the structural gene for thiosulfate reductase. Expression of beta-galactosidase in phs::Mu d1(Apr lac) mutants was dependent on anaerobiosis and the presence of reduced sulfur. It was also strongly influenced by carbon source and growth stage. The results are consistent with a model in which the phs gene encodes a regulatory protein essential for the reduction of thiosulfate to hydrogen sulfide.     

Exogenous hormone applications at pollination for in vitro and in vivo production of cotton interspecific hybrids

Interspecific hybridization of cotton (Gossypium) has been assisted by ovule and embryo culture. These culture methods were compared to exogenous hormone applications for efficient plant production from crosses between Upland cotton, G. hirsutum L., as the maternal parent, and various diploid and tetraploid wild species as the pollen donor. The best exogenous hormone treatment resulted in an average production of five seeds per boll and 4% boll abscission. Generally, exogenous hormones used with standard hybridization techniques were superior to in vitro methods, but for some crosses, embryo culture following hormone applications was warranted.Abbreviations GA gibberellic acid, >90% A₃ - NAA 1-naphthaleneacetic acid - NOA 2-naphthoxyacetic acid.     

The response of Caenorhabditis elegans to hydrogen sulfide and hydrogen cyanide

Hydrogen sulfide (H2S), an endogenously produced small molecule, protects animals from various stresses. Recent studies demonstrate that animals exposed to H2S are long lived, resistant to hypoxia, and resistant to ischemia-reperfusion injury. We performed a forward genetic screen to gain insights into the molecular mechanisms Caenorhabditis elegans uses to appropriately respond to H2S. At least two distinct pathways appear to be important for this response, including the H2S-oxidation pathway and the hydrogen cyanide (HCN)-assimilation pathway. The H2S-oxidation pathway requires two distinct enzymes important for the oxidation of H2S: the sulfide:quinone reductase sqrd-1 and the dioxygenase ethe-1. The HCN-assimilation pathway requires the cysteine synthase homologs cysl-1 and cysl-2. A low dose of either H2S or HCN can activate hypoxia-inducible factor 1 (HIF-1), which is required for C. elegans to respond to either gas. sqrd-1 and cysl-2 represent the entry points in the H2S-oxidation and HCN-assimilation pathways, respectively, and expression of both of these enzymes is highly induced by HIF-1 in response to both H2S and HCN. In addition to their role in appropriately responding to H2S and HCN, we found that cysl-1 and cysl-2 are both essential mediators of innate immunity against fast paralytic killing by Pseudomonas. Furthermore, in agreement with these data, we showed that growing worms in the presence of H2S is sufficient to confer resistance to Pseudomonas fast paralytic killing. Our results suggest the hypoxia-independent hif-1 response in C. elegans evolved to respond to the naturally occurring small molecules H2S and HCN.